You-yuan Wang

The pivotal role of c-Jun NH2-terminal kinase-mediated Beclin 1 expression during anticancer agents-induced autophagy in cancer cells

The c-Jun NH2-terminal kinase (JNK) pathway represents one subgroup of MAP kinases that are activated primarily by cytokines and exposure to environmental stress. Autophagy is a protein-degradation system characterized by the formation of double-membrane vacuoles termed autophagosomes. Autophagy-related gene beclin 1 plays a key role in autophagosome formation. However, the relationships between activation of JNK pathway, autophagy induction and Beclin 1 expression remain elusive. In this study, we used human cancer cell lines CNE2 and Hep3B to investigate the role of JNK-mediated Beclin 1 expression in ceramide-induced autophagic cell death. Ceramide-treated cells exhibited the characteristics of autophagy (that is, acidic vesicular organelle formation and the LC3-II generation). JNK was activated in these two cell lines exposed to ceramide and the phosphorylation of c-Jun also increased. In the meantime, we found that ceramide upregulated Beclin 1 expression in cancer cells. The upregulation of Beclin 1 expression could be blocked by SP600125 (a specific inhibitor of JNK) or a small interfering RNA (siRNA) directed against JNK1/2 or c-Jun. Chromatin immunoprecipitation and luciferase reporter analysis revealed that c-Jun was involved in the regulation of beclin 1 transcription in response to ceramide treatment. In addition, inhibition of JNK activity by SP600125 could inhibit autophagy induction by ceramide. Furthermore, Beclin 1 knockdown by siRNA also inhibited ceramide-mediated autophagic cell death. JNK-mediated Beclin 1 expression was also observed in topotecan-induced autophagy. These data suggest that activation of JNK pathway can mediate Beclin 1 expression, which plays a key role in autophagic cell death in cancer cells.

Acetylcholinesterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis

It has been shown that acetylcholinesterase (AChE) expression was induced during apoptosis and the anti-sense oligonucleotides and siRNA of AChE may prevent apoptosis in various cell types. However, the mechanisms underlying AChE upregulation remain elusive. We demonstrated here that c-Jun NH2-terminal kinase (JNK) could mediate AChE expression. In this study, both etoposide and excisanin A, two anticancer agents, induced apoptosis in colon cancer cell line SW620 as determined by Annexin V staining, the cleavage of caspase-3 and the proteolytic degradation of poly (ADP-ribose) polymerase (PARP). The results showed that both the agents upregulated AChE in SW620 cells. In the meantime, JNK was also activated and the expression and phosphorylation of c-Jun increased in SW620 cells exposed to the two agents. The induced AChE mRNA and protein expression could be blocked by SP600125, a specific inhibitor of SAPK/JNK, and small interfering RNA directed against JNK1/2. Transfection with adenovirus-mediated dominant negative c-Jun also blocked the upregulation of AChE expression. Together, these results suggest that AChE expression may be mediated by the activation of JNK pathway during apoptosis through a c-Jun-dependent mechanism.

A review of clinical and histological parameters associated with contralateral neck metastases in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) has a high incidence of cervical micrometastases and sometimes metastasizes contralaterally because of the rich lymphatic intercommunications relative to submucosal plexus of oral cavity that freely communicate across the midline, and it can facilitate the spread of neoplastic cells to any area of the neck consequently. Clinical and histopathologic factors continue to provide predictive information to contralateral neck metastases (CLNM) in OSCC, which determine prophylactic and adjuvant treatments for an individual patient. This review describes the predictive value of clinical‐histopathologic factors, which relate to primary tumor and cervical lymph nodes, and surgical dissection and adjuvant treatments. In addition, the indications for elective contralateral neck dissection and adjuvant radiotherapy (aRT) and strategies for follow‐up are offered, which is strongly focused by clinicians to prevent later CLNM and poor prognosis subsequently.

miR‐639 regulates transforming growth factor beta‐induced epithelial–mesenchymal transition in human tongue cancer cells by targeting FOXC1

Epithelial‐to‐mesenchymal transition (EMT) is implicated in embryonic development and various pathological events. Transforming growth factor beta (TGFβ) has been reported to induce EMT in tumor cells, which is a critical step in the process of metastasis leading to cancer spreading and treatment failure. However, the involvement of microRNA during the EMT process in tongue squamous cell carcinoma (TSCC) remains to be determined. To address this question, TSCC cell lines SCC9 and CAL27 were treated with human recombinant TGFβ1 for 48 h. miRNA microarray illustrated that miR‐639 was significantly downregulated in TGFβ‐treated SCC9 cells. Ectopic expression of miR‐639 with miRNA mimics effectively blocked TGFβ‐induced EMT in SCC9 and CAL27 cells, but inhibition of miR‐639 in SCC9 and CAL27 cells with antisense oligonucleotides induced EMT. Computational microRNA target predictions detected a conserved sequence matching to the seed region of miR‐639 in the 3′‐UTR of FOXC1 mRNA. Luciferase reporter assays revealed that miR‐639 targets FOXC1. Ectopic expression of FOXC1 induces EMT in TSCC cells. Silencing FOXC1 expression blocked TGFβ‐induced EMT in SCC9 cells. Clinically, reduced miR‐639 expression was associated with metastasis in TSCC and poor patient survival. The data from the present study suggest that reduced expression of miR‐639 underscores the mechanism of TGFβ‐induced EMT in TSCC by targeting FOXC1 and may serve as therapeutic targets in the process of metastasis.

MiR-320a acts as a prognostic factor and Inhibits metastasis of salivary adenoid cystic carcinoma by targeting ITGB3

BackgroundSalivary Adenoid cystic carcinoma (SACC) patients with local invasion and lung metastasis are often resistant to conventional therapy such as operation, chemotherapy and radiotherapy. To explore the underling mechanisms, we studied the roles of miRNA in regulating invasiveness of SACC cells.MethodsMicroRNA profiling was done in SACC cells with microarray. MiRNA mimics or antisense oligonucleotide was transfected and invasiveness of SACC cells was evaluated by adhesion assay and transwell assay. The target gene of miRNA was identified by luciferase reporter assay and “rescue” experiment. Tumor metastasis was evaluated by BALB/c-nu mice xenografts. MiRNA and its target gene expression were identified by in-situ hybridization and immunohistochemistry respectively, in 302 patients from affiliated hospitals of Sun Yat-sen University and in 148 patients from affiliated hospitals of Central South University, and correlated to the clinicopathological status of the patients.ResultsMiR-320a was down-regulated in high lung metastatic ACCM and SACC-LM cells compared with the corresponding low metastatic ACC2 and SACC-83 cells, and inhibited adhesion, invasion and migration of SACC cells by targeting integrin beta 3 (ITGB3). In vivo, enforced miR-320a expression suppressed metastasis of SACC xenografts. In the two independent sets, miR-320a was downregulated in primary SACCs with metastasis compared to those without metastasis, and low expression of this miRNA predicts poor patient survival and rapid metastasis. Multivariate analysis showed that miR-320a expression was an independent indicator of lung metastasis.ConclusionsMiR-320a inhibits metastasis in SACCs by targeting ITGB3 and may serve as a therapeutic target and prognostic marker in salivary cancers.

miR-483-5p determines mitochondrial fission and cisplatin sensitivity in tongue squamous cell carcinoma by targeting FIS1

Mitochondria play an important role in the initiation of apoptosis. However, whether cisplatin can induce apoptosis by initiating a mitochondrial fission pathway and the mechanism underlying this effect remain poorly understood. In this study, we show that the mitochondrial fission protein FIS1 is upregulated upon cisplatin treatment in tongue squamous cell carcinoma (TSCC) cells. FIS1 knockdown can attenuate mitochondrial fission and cisplatin sensitivity. We found that FIS1 is a direct target of miR-483-5p and that miR-483-5p can inhibit mitochondrial fission and cisplatin sensitivity in vitro and in vivo. Furthermore, we found that miR-483-5p and FIS1 are significantly associated with cisplatin sensitivity and with overall survival in patients with TSCC in a retrospective analysis of multiple centers. This study revealed that a novel mitochondrial fission pathway composed of miR-483-5p and FIS1 regulates cisplatin sensitivity. The modulation of miR-483-5p and FIS1 levels may provide a new approach for increasing cisplatin sensitivity.

A role for cancer-associated fibroblasts in inducing the epithelial-to-mesenchymal transition in human tongue squamous cell carcinoma

OBJECTIVES Lymph node metastasis is a prominent clinical feature of tongue squamous cell carcinoma (TSCC) and is associated with a higher mortality rate. Carcinoma-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME), play an important role in tumor progression, and are associated with a poor prognosis. The aim of this study was to examine the role of CAFs in promoting the invasion of TSCC through the epithelial-to-mesenchymal transition (EMT). MATERIALS AND METHODS A series of matched CAF and normal fibroblast (NF) pairs were assessed for cell morphology and for the expression of alpha smooth muscle actin (α-SMA), stromal cell-derived factor-1 (SDF1), fibroblast-activating protein (FAP), vimentin, and cytokeratin (CK) markers. Transwell assays, Western blot analysis, reverse transcription-PCR, and immunofluorescence staining were used to assess the role of CAFs, as compared to that of NFs, in promoting proliferation, migration, invasion, and EMT in TSCC. RESULTS Both CAF and NF primary cultures expressed vimentin but not CK. CAFs showed significantly higher α-SMA protein levels, SDF1 secretion, and mRNA levels of α-SMA, SDF1, and FAP. We also found that co-culture with CAFs enhanced the proliferation and invasion of SCC9 cells. Moreover, co-culture with CAFs induced upregulation of the EMT markers fibronectin and vimentin, downregulation of E-cadherin, and enhanced invasion in SCC9 cells. CONCLUSION These results suggest that CAFs induce EMT marker expression and functional changes in TSCCs.

Overexpression of CD147 contributes to the chemoresistance of head and neck squamous cell carcinoma cells.

Head and neck cancer is the sixth most common cancer in the world and most of them are squamous cell carcinomas. High frequency of cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) leads to tumor relapse and irresponsiveness to cisplatin-based chemotherapy. However, the mechanisms underlying cisplatin resistance is still largely unrevealed. In this study, we found that CD147 was overexpressed in cisplatin-resistant HNSCC cell lines. Based on the result, CD147 expression was down-regulated in the cisplatin-resistant cell line and we observed that the sensitivity to cisplatin increased, as showed in the results of MTT assay and PI-staining apoptotic detection. Meanwhile, transfection of CD147 expression vector promoted the occurrence of cisplatin resistance in the cisplatin-sensitive cell line. Simultaneously blocking of uPAR with neutralizing antibody would significantly prevent the occurrence of cisplatin resistance induced by CD147 overexpression. In conclusion, our study finds that CD147 is also involved in mediating cisplatin resistance in HNSCC and uPAR serves as a possible candidate that collaborates with CD147 in this process.

SMAS fold flap and ADM repair of the parotid bed following removal of parotid haemangiomas via pre- and retroauricular incisions to improve cosmetic outcome and prevent Frey's syndrome

The growth of parotid haemangiomas during the proliferative phase may be rapid and unpredictable. Involution often takes many years, with attendant psychological sequelae to the child. Although conservative management is usually proposed for parotid haemangiomas occurring in infancy, this may not be particularly helpful and the haemangioma difficult to conceal. The purpose of this study was to evaluate the reliable and aesthetic benefit of using a superficial musculoaponeurotic system (SMAS) fold flap and allograft dermal matrix (ADM) repair of the parotid bed following parotid haemangiomas via pre- and retroauricular incision. Forty-three paediatric patients (33 boys and 10 girls) with haemangiomas involving the parotid gland underwent total parotidectomy using a pre- and retroauricular approach with intraoperative placement of ADM within the parotid bed. They further underwent repair of the parotid bed with SMAS fold flaps. A panel of three plastic surgeons assessed the cosmetic outcomes. All of the patients were evaluated using a short questionnaire; postoperative gustatory sweating was assessed using a modification of Minors starch-iodine test.

Disrupting G6PD-mediated Redox homeostasis enhances chemosensitivity in colorectal cancer

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that generates NADPH to maintain reduced glutathione (GSH), which scavenges reactive oxygen species (ROS) to protect cancer cell from oxidative damage. In this study, we mainly investigate the potential roles of G6PD in colorectal cancer (CRC) development and chemoresistance. We discover that G6PD is overexpressed in CRC cells and patient specimens. High expression of G6PD predicts poor prognosis and correlated with poor outcome of oxaliplatin-based first-line chemotherapy in patients with CRC. Suppressing G6PD decreases NADPH production, lowers GSH levels, impairs the ability to scavenge ROS levels, and enhances oxaliplatin-induced apoptosis in CRC via ROS-mediated damage in vitro. In vivo experiments further shows that silencing G6PD with lentivirus or non-viral gene delivery vector enhances oxaliplatin anti-tumor effects in cell based xenografts and PDX models. In summary, our finding indicated that disrupting G6PD-mediated NADPH homeostasis enhances oxaliplatin-induced apoptosis in CRC through redox modulation. Thus, this study indicates that G6PD is a potential prognostic biomarker and a promising target for CRC therapy.